Mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eIF2alpha phosphorylation and PKR

In response to mammalian orthoreovirus (MRV) infection, cells initiate a stress response that includes eIF2α phosphorylation and protein synthesis inhibition. We have previously shown that early in infection, MRV activation of eIF2α phosphorylation results in the formation of cellular stress granules (SGs). In this work, we show that as infection proceeds, MRV disrupts SGs despite sustained levels of phosphorylated eIF2α and, further, interferes with the induction of SGs by other stress inducers. MRV interference with SG formation occurs downstream of eIF2α phosphorylation, suggesting the virus uncouples the cellular stress signaling machinery from SG formation. We additionally examined mRNA translation in the presence of SGs induced by eIF2α phosphorylation-dependent and -independent mechanisms. We found that irrespective of eIF2α phosphorylation status, the presence of SGs in cells correlated with inhibition of viral and cellular translation. In contrast, MRV disruption of SGs correlated with the release of viral mRNAs from translational inhibition, even in the presence of phosphorylated eIF2α. Viral mRNAs were also translated in the presence of phosphorylated eIF2α in PKR(-/-) cells. These results suggest that MRV escape from host cell translational shutoff correlates with virus-induced SG disruption and occurs in the presence of phosphorylated eIF2α in a PKR-independent manner.     

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2α phosphorylation-dependent and -independent pathways that target translation initiation.     

Resistance to vesicular stomatitis virus infection requires a functional cross talk between the eukaryotic translation initiation factor 2alpha kinases PERK and PKR

Phosphorylation of the alpha (alpha) subunit of the eukaryotic translation initiation factor 2 (eIF2) leads to the inhibition of protein synthesis in response to diverse stress conditions, including viral infection. The eIF2alpha kinase PKR has been shown to play an essential role against vesicular stomatitis virus (VSV) infection. We demonstrate here that another eIF2alpha kinase, the endoplasmic reticulum-resident protein kinase PERK, contributes to cellular resistance to VSV infection. We demonstrate that mouse embryonic fibroblasts (MEFs) from PERK(-/-) mice are more susceptible to VSV-mediated apoptosis than PERK(+/+) MEFs. The higher replication capacity of VSV in PERK(-/-) MEFs results from their inability to attenuate viral protein synthesis due to an impaired eIF2alpha phosphorylation. We also show that VSV-infected PERK(-/-) MEFs are unable to fully activate PKR, suggesting a cross talk between the two eIF2alpha kinases in virus-infected cells. These findings further implicate PERK in virus infection, and provide evidence that the antiviral and antiapoptotic roles of PERK are mediated, at least in part, via the activation of PKR.     

Influenza A Virus Host Shutoff Disables Antiviral Stress-Induced Translation Arrest

Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication.     

Stress granule assembly is mediated by prion-like aggregation of TIA-1

TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in cells subjected to environmental stress. The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD). Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level-expressing cells but prevent SG assembly in high-level-expressing cells. The PRD of TIA-1 exhibits many characteristics of prions: concentration-dependent aggregation that is inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of HSP70, a feedback regulator of PRD disaggregation. Substitution of the PRD with the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly, confirming that a prion domain can mediate the assembly of SGs. Mouse embryomic fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2alpha in response to arsenite. Our results reveal that prion-like aggregation of TIA-1 regulates SG formation downstream of eIF2alpha phosphorylation in response to stress.     

Hydrogen peroxide induces stress granule formation independent of eIF2α phosphorylation

In cells exposed to environmental stress, inhibition of translation initiation conserves energy for the repair of cellular damage. Untranslated mRNAs that accumulate in these cells move to discrete cytoplasmic foci known as stress granules (SGs). The assembly of SGs helps cells to survive under adverse environmental conditions. We have analyzed the mechanism by which hydrogen peroxide (H(2)O(2))-induced oxidative stress inhibits translation initiation and induces SG assembly in mammalian cells. Our data indicate that H(2)O(2) inhibits translation and induces the assembly of SGs. The assembly of H(2)O(2)-induced SGs is independent of the phosphorylation of eIF2α, a major trigger of SG assembly, but requires remodeling of the cap-binding eIF4F complex. Moreover, H(2)O(2)-induced SGs are compositionally distinct from canonical SGs, and targeted knockdown of eIF4E, a protein required for canonical translation initiation, inhibits H(2)O(2)-induced SG assembly. Our data reveal new aspects of translational regulation induced by oxidative insults.     

A Highlights from MBoC Selection: Translation suppression promotes stress granule formation and cell survival in response to cold shock

Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2α (eIF2α) and the mTOR pathway. Here we identify cold shock as a novel trigger of SG assembly in yeast and mammals. Whereas cold shock–induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures. Cold shock causes eIF2α phosphorylation through the kinase PERK in mammalian cells, yet this pathway is not alone responsible for translation arrest and SG formation. In addition, cold shock leads to reduced mitochondrial function, energy depletion, concomitant activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR signaling. Compound C, a pharmacological inhibitor of AMPK, prevents the formation of SGs and strongly reduces cellular survival in a translation-dependent manner. Our results demonstrate that cells actively suppress protein synthesis by parallel pathways, which induce SG formation and ensure cellular survival during hypothermia.     

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.     

Modulation of stress granules and P bodies during dicistrovirus infection

Stress granules (SGs) are dynamic cytosolic aggregates composed of ribonucleoproteins that are induced during cellular stress when protein synthesis is inhibited. The function of SGs is poorly understood, but they are thought to be sites for reorganizing mRNA and protein. Several viruses can modulate SG formation, suggesting that SGs have an impact on virus infection. In this study, we have investigated the relationship of SG formation in Drosophila S2 cells infected by cricket paralysis virus (CrPV), a member of the Dicistroviridae family. Despite a rapid shutoff of host translation during CrPV infection, several hallmark SG markers such as the Drosophila TIA-1 and G3BP (RasGAP-SH3-binding protein) homologs, Rox8 and Rin, respectively, do not aggregate in CrPV-infected cells, even when challenged with potent SG inducers such as heat shock, oxidative stress, and pateamine A treatment. Furthermore, we demonstrate that a subset of P body markers become moderately dispersed at late times of infection. In contrast, as shown by fluorescent in situ hybridization, poly(A)(+) RNA granules still form at late times of infection. These poly(A)(+) RNA granules do not contain viral RNA nor do they colocalize with P body markers. Finally, our results demonstrate that the CrPV viral 3C protease is sequestered to SGs under cellular stress but not during virus infection. In summary, we propose that dicistrovirus infection leads to the selective inhibition of distinct SGs so that viral proteins are available for viral processing.     

Dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection

Virus infection-induced global protein synthesis suppression is linked to assembly of stress granules (SGs), cytosolic aggregates of stalled translation preinitiation complexes. To study long-term stress responses, we developed an imaging approach for extended observation and analysis of SG dynamics during persistent hepatitis C virus (HCV) infection. In combination with type 1 interferon, HCV infection induces highly dynamic assembly/disassembly of cytoplasmic SGs, concomitant with phases of active and stalled translation, delayed cell division, and prolonged cell survival. Double-stranded RNA (dsRNA), independent of viral replication, is sufficient to trigger these oscillations. Translation initiation factor eIF2α phosphorylation by protein kinase R mediates SG formation and translation arrest. This is antagonized by the upregulation of GADD34, the regulatory subunit of protein phosphatase 1 dephosphorylating eIF2α. Stress response oscillation is a general mechanism to prevent long-lasting translation repression and a conserved host cell reaction to multiple RNA viruses, which HCV may exploit to establish persistence.     

Evidence That Ternary Complex (eIF2-GTP-tRNAiMet)–Deficient Preinitiation Complexes Are Core Constituents of Mammalian Stress Granules

Environmental stress-induced phosphorylation of eIF2alpha inhibits protein translation by reducing the availability of eIF2-GTP-tRNA(i)Met, the ternary complex that joins initiator tRNA(Met) to the 43S preinitiation complex. The resulting untranslated mRNA is dynamically routed to discrete cytoplasmic foci known as stress granules (SGs), a process requiring the related RNA-binding proteins TIA-1 and TIAR. SGs appear to be in equilibrium with polysomes, but the nature of this relationship is obscure. We now show that most components of the 48S preinitiation complex (i.e., small, but not large, ribosomal subunits, eIF3, eIF4E, eIF4G) are coordinately recruited to SGs in arsenite-stressed cells. In contrast, eIF2 is not a component of newly assembled SGs. Cells expressing a phosphomimetic mutant (S51D) of eIF2alpha assemble SGs of similar composition, confirming that the recruitment of these factors is a direct consequence of blocked translational initiation and not due to other effects of arsenite. Surprisingly, phospho-eIF2alpha is recruited to SGs that are disassembling in cells recovering from arsenite-induced stress. We discuss these results in the context of a translational checkpoint model wherein TIA and eIF2 are functional antagonists of translational initiation, and in which lack of ternary complex drives SG assembly.     

Monocyte chemotactic protein-induced protein 1 (MCPIP1) suppresses stress granule formation and determines apoptosis under stress

It is unclear how stress granule (SG) formation and cellular apoptosis are coordinately regulated. MCPIP1 (monocyte chemotactic protein-induced protein 1), also known as Zc3h12a, is a critical regulator of the inflammatory response and immune homeostasis. However, the role of MCPIP1 in stress response remains unknown. Here, we report that overexpression of MCPIP1 inhibited the assembly of SGs in response to various stresses. Conversely, MCPIP1-deficient splenocytes developed more SGs even without stress. On the other hand, overexpression of MCPIP1 sensitized RAW 264.7 cells to apoptosis under stress, whereas MCPIP1-deficient cells were resistant to stress-induced apoptosis. Mutagenesis study showed that the ability of MCPIP1 to repress SG formation is dependent on its deubiquitinating activity. Consistently, MCPIP1 negatively regulated stress-induced phosphorylation of eIF2α and thus released stress-induced inhibition of protein translation. However, MCPIP1 also inhibited 15-deoxy-Δ(12,14)-prostaglandin J(2)-induced SG formation, which was reported to be independent of eIF2α phosphorylation. Taken together, these results suggest that MCPIP1 coordinates SG formation and apoptosis during cellular stress and may play a critical role in immune homeostasis and resolution of macrophage inflammation.     

A novel function of eIF2alpha kinases as inducers of the phosphoinositide-3 kinase signaling pathway

Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the alpha subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2alpha kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR(-/-) or PERK(-/-) mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2alpha kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2alpha phosphorylation as demonstrated by the inactivation of endogenous eIF2alpha by small interfering RNA or utilization of MEFs bearing the eIF2alpha Ser51Ala mutation. Our data reveal a novel property of eIF2alpha kinases as activators of PI3K signaling and cell survival.     

Herpes simplex virus 2 infection impacts stress granule accumulation

Interference with stress granule (SG) accumulation is gaining increased appreciation as a common strategy used by diverse viruses to facilitate their replication and to cope with translational arrest. Here, we examined the impact of infection by herpes simplex virus 2 (HSV-2) on SG accumulation by monitoring the localization of the SG components T cell internal antigen 1 (TIA-1), Ras-GTPase-activating SH3-domain-binding protein (G3BP), and poly(A)-binding protein (PABP). Our results indicate that SGs do not accumulate in HSV-2-infected cells and that HSV-2 can interfere with arsenite-induced SG accumulation early after infection. Surprisingly, SG accumulation was inhibited despite increased phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), implying that HSV-2 encodes previously unrecognized activities designed to maintain translation initiation downstream of eIF2α. SG accumulation was not inhibited in HSV-2-infected cells treated with pateamine A, an inducer that works independently of eIF2α phosphorylation. The SGs that accumulated following pateamine A treatment of infected cells contained G3BP and PABP but were largely devoid of TIA-1. We also identified novel nuclear structures containing TIA-1 that form late in infection. These structures contain the RNA binding protein 68-kDa Src-associated in mitosis (Sam68) and were noticeably absent in infected cells treated with inhibitors of viral DNA replication, suggesting that they arise as a result of late events in the virus replicative cycle.     

Inhibition of the ubiquitin-proteasome system induces stress granule formation

The inhibition of the ubiquitin-dependent proteasome system (UPS) via specific drugs is one type of approach used to combat cancer. Although it has been suggested that UPS inhibition prevents the rapid decay of AU-rich element (ARE)-containing messages, very little is known about the cellular mechanisms leading to this effect. Here we establish a link between the inhibition of UPS activity, the formation of cytoplasmic stress granules (SGs), and mRNA metabolism. The assembly of the SGs requires the phosphorylation of the translation initiation factor eIF2alpha by a mechanism involving the stress kinase GCN2. On prolonged UPS inhibition and despite the maintenance of eIF2alpha phosphorylation, SGs disassemble and translation recovers in an Hsp72 protein-dependent manner. The formation of these SGs coincides with the disassembly of processing bodies (PBs), known as mRNA decay entities. As soon as the SGs assemble, they recruit ARE-containing messages such as p21(cip1) mRNA, which are stabilized under these conditions. Hence, our findings suggest that SGs could be considered as one of the players that mediate the early response of the cell to proteasome inhibitors by interfering temporarily with mRNA decay pathways.     

Selenite targets eIF4E-binding protein-1 to inhibit translation initiation and induce the assembly of non-canonical stress granules

Stress granules (SGs) are large cytoplasmic ribonucleoprotein complexes that are assembled when cells are exposed to stress. SGs promote the survival of stressed cells by contributing to the reprogramming of protein expression as well as by blocking pro-apoptotic signaling cascades. These cytoprotective effects implicated SGs in the resistance of cancer cells to radiation and chemotherapy. We have found that sodium selenite, a selenium compound with chemotherapeutic potential, is a potent inducer of SG assembly. Selenite-induced SGs differ from canonical mammalian SGs in their morphology, composition and mechanism of assembly. Their assembly is induced primarily by eIF4E-binding protein1 (4EBP1)-mediated inhibition of translation initiation, which is reinforced by concurrent phosphorylation of eIF2α. Selenite-induced SGs lack several classical SG components, including proteins that contribute to pro-survival functions of canonical SGs. Our results reveal a new mechanism of mammalian SG assembly and provide insights into how selenite cytotoxicity may be exploited as an anti-neoplastic therapy.     

Inhibition of host and viral translation during vesicular stomatitis virus infection. eIF2 is responsible for the inhibition of viral but not host translation

In cells that allow replication of vesicular stomatitis virus (VSV), there are two phases of translation inhibition: an early block of host translation and a later inhibition of viral translation. We investigated the phosphorylation of the alpha subunit of the eIF2 complex during these two phases of viral infection. In VSV-infected cells, the accumulation of phosphorylated (inactivated) eIF2alpha did not begin until well after host protein synthesis was inhibited, suggesting that it only plays a role in blocking viral translation later after infection. Consistent with this, cells expressing an unphosphorylatable eIF2alpha showed prolonged viral protein synthesis without an effect on host protein synthesis inhibition. Induction of eIF2alpha phosphorylation at early times of viral infection by treatment with thapsigargin showed that virus and host translation are similarly inhibited, demonstrating that viral and host messages are similarly sensitive to eIF2alpha phosphorylation. A recombinant virus that expresses a mutant matrix protein and is defective in the inhibition of host and virus protein synthesis showed an altered phosphorylation of eIF2alpha, demonstrating an involvement of viral protein function in inducing this antiviral response. This analysis of eIF2alpha phosphorylation, coupled with earlier findings that the eIF4F complex is modified earlier during VSV infection, supports a temporal/kinetic model of translation control, where at times soon after infection, changes in the eIF4F complex result in the inhibition of host protein synthesis; at later times, inactivation of the eIF2 complex blocks VSV protein synthesis.     

Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation

Eukaryotic cells express a family of eukaryotic translation initiation factor 2 alpha (eIF2alpha) kinases (eg, PKR, PERK-PEK, GCN2, HRI) that are individually activated in response to distinct types of environmental stress. Phosphorylation of eIF2alpha by one or more of these kinases reduces the concentration of eIF2-guanosine triphosphate (GTP)-transfer ribonucleic acid for methionine (tRNA(Met)), the ternary complex that loads tRNA(Met) onto the small ribosomal subunit to initiate protein translation. When ternary complex levels are reduced, the related RNA-binding proteins TIA-1 and TIAR promote the assembly of a noncanonical preinitiation complex that lacks eIF2-GTP-tRNA(Met). The TIA proteins dynamically sort these translationally incompetent preinitiation complexes into discrete cytoplasmic domains known as stress granules (SGs). RNA-binding proteins that stabilize or destabilize messenger RNA (mRNA) are also recruited to SGs during stress. Thus, TIA-1 and TIAR act downstream of eIF2alpha phosphorylation to promote SG assembly and facilitate mRNA triage during stress. The role of the SG in the integration of translational efficiency, mRNA stability, and the stress response is discussed.